The present invention relates to the use of nucleic acid analogues in blocking nucleic acid amplification procedures and to diagnostic/analytical techniques based thereon.
Nucleic acid amplification techniques are now in widespread use. These include the "PCR" (polymerase chain reaction) procedures described in EP-A-0200362 and EP-A-0201184 which is the technique in most widespread use, but also the "LCR" (ligase chain reaction) described in EP-A-0320308 and the so-called "NASBA" or "3SR" technique which is described in "Proc. Natl. Acad. Sci. USA" Vol 87. pp 1874-1878 March 1990 and "Nature" Vol 350, No 6313. pp 91-92 7th March 1991.
A major problem in the use of these procedures in diagnostics is the production of false positives by the carry over of amplified nucleic acid sequences from previous reactions. As these procedures are capable of producing a positive result if even a single molecule containing the target sequence is present, it is of course very easy for such a false positive to occur. At present it is necessary for the reaction product of such procedures to be worked up before the success or failure of the amplification procedure can be determined. The involves contact between the reaction product and several pieces of laboratory equipment such as pipettes, as well as with personnel which can lead to traces of the amplification product being available to contaminate future runs.
Much effort is currently going into developing so called "intrinsic" procedures in which the success of the amplification reaction can be monitored without any handling of the amplification product and in an unopened reaction vessel.
New forms of nucleic acid analogue are described in Patent Co-operation Treaty application. No. PCT/EP92/01220 filed on 22nd May 1992 which selectively bind conventional nucleic acids of complementary sequence to form hybrids which are more stable against dehybridisation by heat than are similar hybrids between conventional nucleic acids. We have now found that it is possible to exploit this greater hybrid stability to block selectively nucleic acid amplification procedures. This technique may be used to prevent false positives in such amplification procedures. It may also be exploited as an essential part of certain diagnostics/analytical approaches.
In a first aspect the invention provides a method of inhibiting a nucleic acid amplification procedure comprising providing in said procedure an effective amount of a nucleic acid analogue sufficiently complementary to a sequence of nucleic acid participating in an essential manner in said amplification procedure to hybridises to said sequence sufficiently strongly to prevent the effective participation of said sequence in the amplification procedure. Thus this aspect of the invention includes a method of inhibiting a nucleic acid amplification procedure in which procedure each strand of a double stranded target nucleic acid has a region used as a template for one or more primers which is or are extended or linked to form a second template complementary to said template, wherein a nucleic acid analogue sufficiently complementary to a sequence of a said template or a said primer to hybridise therewith is provided and wherein said nucleic acid analogue hybridises sufficiently strongly to the respective template or primer to block primer template hybridisation or to block primer extension or primer linking under the conditions of the procedure.
Preferably, said procedure is a PCR or LCR or a 3SR procedure. "Anchored" and "inverted" PCR procedures are included.
The "double stranded target nucleic acid" which has in each strand a region acting as a template is not necessarily a starting material, nor need it necessarily be the direct object of desired amplification in the procedure.
In PCR the "double stranded target nucleic acid" will usually be incorporated in the starting material and it will generally be the amplification of at least one strand thereof which is desired. However, one can start a PCR reaction with only one DNA strand, the complementary strand being produced in the first cycle of primer extension. In later cycles, the "double stranded target nucleic acid" will be composed of products from previous cycles predominantly.
In LCR, the "double stranded target nucleic acid" will similarly initially normally be incorporated in the starting material and in later cycles will predominantly be the amplification product.
It is not necessarily the case that primers are hybridised to each strand in the same part of the amplification cycle or, indeed only after the double stranded target nucleic acid has been formed. Thus in the 3SR technique, a starting RNA strand is hybridised to a first primer which is extended as a complementary DNA strand by reverse transcriptase to form the "double stranded target nucleic acid" referred to. After destruction of the hybridised RNA strand by RNase H, a second primer is hybridised to the DNA strand and extended by reverse transcriptase to form a double stranded DNA molecule. Thus by this stage, both strands present in the DNA/RNA will have in their time been hybridised to a primer which has then been extended.
The first primer is so constructed that the DNA/DNA product includes a promoter for an RNA polymerase such as T7 RNA polymerase which using the DNA/DNA product as a template produces multiple RNA copies corresponding to the RNA starting material, which each in turn become starting materials for the first primer and reverse transcriptase to act upon.
Said amplification procedure may be conducted in the absence of said nucleic acid analogue to build-up a desired level of amplification product and said nucleic acid analogue may then be added.
Preferably the said nucleic acid analogue hybridises to said amplification product. This will shut down the amplification and can leave the products in the form of stable nucleic acid--nucleic acid analogue hybrids which are unable to act as templates if they contaminate a subsequent reaction. "Open" rather than "intrinsic" procedures are thereby rendered safe against carry over contamination.
Alternatively or additionally, apparatus to be used in an amplification procedure of said type can be treated with a solution containing said nucleic acid analogue to hybridise said analogue to any amplification product which may be present as a contamination, and the apparatus may be thereafter washed and used in a said amplification procedure, amplification in said procedure of any amplification product present initially as a contamination in said apparatus being prevented by said hybridisation to said analogue.
The invention includes therefore a method of preventing a nucleic acid amplification product from serving as a template in a subsequent amplification procedure, which method comprises hybridising thereto a nucleic acid analogue which forms therewith a hybrid which is stable under the conditions of the subsequent amplification procedure.
The invention also includes a method of preventing any nucleic acid amplification product which may be present in an environment from serving as a contaminating template in a subsequent amplification procedure for the nucleic acid, which method comprises treating the environment with a nucleic acid analogue which forms with said amplification product a hybrid which is stable under the conditions of said subsequent amplification procedure.
In practice, one may wash all or some equipment to be utilised in the procedure with a solution containing the nucleic acid analogue, e.g. in a hybridisation buffer, so as to form a stable hybrid, between the analogue and any nucleic acid present which contains the target sequence for the amplification. The hybrid formed with the nucleic acid analogue is sufficiently stable to withstand the conditions of the subsequent amplification procedure and the contaminant target sequence is thereby prevented from participating in such a procedure to produce a false positive.
The nucleic acid analogue for use in this and other aspects of the invention preferably contains from five to 20 nucleobase binding ligands e.g. from ten to fifteen ligands.
One may provide a nucleic acid analogue which is tailored to halt amplification of a specific nucleic acid sequence or one may provide a reagent containing a mixture of nucleic acid analogue sequences which will stop a number of different amplifications. Preferably, a reagent for this latter purpose contains a multiplicity or multitude of sequences. In the extreme, one may synthesise nucleic acid analogues using a mixture of monomer synthons (including ligands, complementary to each of the four DNA bases) in each step so as to construct molecules each containing a random sequence of ligands. A sufficient quantity of such a reagent should contain a sufficient number of molecules complementary to any amplification product to be able to inhibit any amplification procedure.
Generally, it will be preferred for each molecule of nucleic acid analogue in such a reagent to be at least a 10-mer, more preferably at least a 12-mer, e.g. a 15- to 20-mer.
The selective blocking of amplification procedures may be put to work more actively as part of a diagnostic process.
At present, when using methods such as PCR to amplify a sequence with a view to determining whether a particular sequence is present, such as a single base change from an alternative sequence (e.g. a single base mutation from the "normal" sequence in a gene), the procedures for detecting the sequence in the amplification product are laborious. These include sequencing the entire product to verify the presence of the altered base. They include also detecting an altered pattern of digestion products produced by one or more restriction enzymes if the change or changes in the gene sequence affect a restriction site. They also include hybridising to the product a labelled nucleic acid probe for the mutated sequence under conditions which are sufficiently stringent to differentiate between the possible sequences.